The present invention relates to multispecific and multivalent antigen-binding polypeptides and methods for producing them.
The ability to target and activate cytotoxic lymphocytes opens the way to utilize natural effector functions to treat cancer, auto-immune disorders and infectious diseases. See, Segal et al., Chem. Immunology, Ed. Ishizaka et al., 47:179-213 (1989). Bispecific antibodies allow this linking of target cell to the effector cell. However, the bispecific antibodies generated so far have two major drawbacks.
Firstly, the specificities against human antigens are predominantly encoded by rodent antibodies and would result in a human anti-murine antibody (HAMA) response in repeated or prolonged use. Although the specificity problem has partially been overcome by forming chimeric (Knox, et al., Blood, 77:20, 1991); Shaw, et al., J. Biol. Response Med., 7:204, (1988); and Oudin, et al., Proc. Natl. Acad. Sci. USA, 63:266, (1971)) and humanized (Hale, et al., Lancet, 2:1394, (1988)) antibodies, the problem of immunogenicity still persists. Advances in applying combinatorial antibody repertoire cloning for the generation of human monoclonal antibodies provides a source of human derived variable, framework and constant regions thus theoretically avoiding a HAMA response. This approach works well when an immune source for the desired specificity is available, but is of limited value in the search for antibodies which specifically bind human cell surface molecules.
Secondly, current methods for producing bispecific molecules rely on either chemical cross linking or heterohybridoma (quadroma) formation. The former approach results in a heteroconjugates resulting in preparations that vary from batch to batch in terms of composition and consequently potency. The latter approach involves fusing the two hybridoma cell lines producing the desired antibodies, giving rise to a quadroma cell encoding and expressing all the H and L chains. The desired H and L chain combination for the bispecific antibody is usually only 15% of the total antibody and is difficult to isolate from the closely related pool of antibodies as described by Milstein et al., Nature, 305:537 (1983).
Ideally human variable regions conferring the desired specificities should be used to construct a single molecule containing both the specificities as the major if not only product. Alternatively, variable regions conferring the desired specificities should be used to construct two polypeptide molecules containing immunologically distinct constant region domains wherein the majority of the two polypeptide molecules are linked by disulphide bonding. Such molecules would avoid the HAMA response while facilitating production of a homogeneous product for characterization and clinical evaluation.
The ability to PCR amplify, directionally clone and express antibody variable regions from cDNA has allowed hybridoma technology to be bypassed. Diverse high affinity antibodies have been generated to hapten, virus particles and protein antigens, thereby recapitulating functional molecules appearing during the natural immune response in animals and in humans. See, for example, Skerra et al., Science, 240:1038-1041 (1988); Better et al., Science, 240:1041-1043 (1988); Orlandi et al., Proc. Natl. Acad. Sci., USA, 86:3833-3837 (1989); Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991); and Barbas et al., Proc. Natl. Acad. Sci.. USA, 88:7978-7982 (1991). Marks et al., J. Mol. Biol., 222:581-597 (1991) demonstrated that active single chain antibody Fv fragments with affinity constants in the range of 106-107 M-l against a hapten or a small number of epitopes on a protein can be obtained directly from non-immune combinatorial immunoglobulin libraries. More recently, a combinatorial library approach was used to select monoclonal antibodies from non-immune mice and subsequently affinity mature the specificities, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predefined specificities and (ii) increasing the affinity of the selected antibodies binding sites by random mutagenesis. See, Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992).
In addition, large libraries of antibody Fab fragments have been displayed on the surface of phage. See, for example, Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991); Hoogenboom et al., Nuc. Acids. Res., 19:4133-4137 (1991); Burton et al., Proc. Natl. Acad. Sci., USA, 88:10134-10137 (1991); Griffiths et al., EMBO J., 12:725-734 (1993); and Soderlind et al., Bio/Technology, 11:503-507 (1993). In essence, the antigen recognition unit has been linked to instructions for its production. An iterative process of mutation followed by selection has also been developed allowing for the rapid generation of specific antibodies from germ line sequences as describe by Gram et al., supra.
The B cell immune response to an antigen can be viewed to occur in two stages. The initial stage generates low affinity antibodies mostly of the IgM isotype from an existing pool of the B-cell repertoire available at the time of immunization. The second stage which is driven by antigen stimulation produces high affinity antibodies predominantly of the IgG isotype, starting with the VH and VL genes selected in the primary response. The predominant mechanism for affinity maturation is hypermutation of variable region genes (and possibly gene conversion) followed by selection of those cells which produce antibodies of the highest affinity. In its simplest form, the initial stage of the immune response can be recreated in vitro by generating a combinatorial library of PCR amplified IgM/G and light chains from the bone marrow of adults. This is a close approximation to the naive, unselected repertoire, since the majority of the B cells in the bone marrow expressing IgM/G chains have not been subjected to tolerance and antigen selection, and should therefore represent all the combinatorial diversity of immunoglobulin V-regions. See, Decker et al., J. Immunol., 146:350-361 (1991). The phagemid pComb8 facilitates the display of multiple copies of the single chain antibody along the phage surface permitting the access to low affinity antibodies as described by Kang et al., supra and Gram et al., supra. Hence, specific VH and VL pairs could possibly be enriched from a diverse naive repertoire.
The second stage of the immune response in vivo involves affinity maturation of the selected specificities by mutation and selection. An efficient way to generate random mutations is by an error-prone replication mechanism, either by targeting the mutations to the antibody binding sites by error-prone PCR as described by Leung et al., J. Methods Cell Molecular Biol., 1:1-15 (1989), or by passaging the phagemid carrying the genetic information for the antigen binding domain through an E.coli mutD strain, in which the spontaneous mutation frequency is 103 to 105 times higher than in a wild-type strain as described by Fowler et al., J. Bacteriol., 167:130-137 (1986). Selected VH and VL pairs could be subjected to error prone PCR (also gene conversion by PCR is feasible) and the resulting products cloned into phagemid pComb3 which facilitates the display of a single copy of the mutant single chain Fv, such low level of display permits the isolation of the highest affinity molecules.
Variations of single chain bispecific molecules have been constructed in bacteria by linking two single chain heavy and light chain variable domains (sFv) with a synthetic linker. See, for example, Wels et al., Bio/Technology, 10:1128-1132 (1992); Stemmer et al., BioTechniques, 14:256-265 (1993); Goshorn et al., Cancer Res., 53:2123-2127 (1993); and Bos et al., Biotherapy, 5:187-199 (1992). The molecules generated, however, are incorrectly folded and on denaturing and refolding result in very low yield. This may be intrinsically due to expressing both the specificities as a single protein giving rise to inter- and intra-molecular heterodimers. Earlier studies have shown that vector-expressed heavy and light chains that were secreted into the periplasmic space assembled with a disulfide bond linking the constant domain. See, Kang et al., supra and Barbas et al., supra.
Bispecific molecules have also been shown to assemble as dimers. Pack et al., Biochem., 31:1579-1584 (1992) have described one such dimeric antibody produced in E. coli that is based on a sFv fragment with a flexible hinge region from mouse IgG3 and an amphipathic helix fused to the carboxy terminus of the antibody fragment. For reviews of protein engineering of antibodies including bivalent and bispecific antibodies see Skerra, Current Opinion in Immunol., 5:256-262 (1993) and Sandhu, Crit. Reviews Biotech., 12:437-462 (1992). While molecules exist that have both bivalent and bispecific properties, these molecules have undesirable properties of being expressed and purified in low quantities, unstable in a dimer conformation, of having regions that would induce an immunogenic response against the molecule or having effector and complement activation functions mediated by the presence of Fc.
Methods have now been discovered to produce bispecific multivalent polypeptides derived from human antibodies thus eliminating an immune response when used in immunotherapy. The bispecific multivalent polypeptides can be expressed and purified to near homogeneity by sequential purification.
Methods have now been discovered that result in the production of molecules that exist in stable multimeric conformations having more than one antigen-binding specificity while lacking undesirable effector or complement activation functions. Because of the multivalent conformations, the compositions of this invention are not destroyed by the body and circulate as functional molecules much like intact immunoglobulins for longer periods of time than their Fab counterparts. The methods of this invention provide for the formation of multimeric compositions in functional form in E. coli and mammalian cells. In addition, the stable multimeric polypeptide molecules exhibit increased avidity over the monomeric antigen binding sites due to the functional folding of the variable domains in the compositions to form two ligand or antigen binding sites. The methods of this invention are useful for generating compositions that have a predetermined immunospecificity that bind to target molecules with increased avidity.
The present invention can be advantageously applied to the production of multivalent molecules of predetermined specificity, i.e., it can be used to produce antibodies, T-cell receptors and the like that bind a preselected ligand.
In one embodiment, the present invention contemplates a composition comprising a bivalent polypeptide having an amino acid residue sequence according to the formula Vxe2x80x94Xxe2x80x94V, wherein V is an antigen binding site, and X is an amino acid residue sequence of from about 5 to about 120 amino acid residues. In preferred embodiments, X is an immunoglobulin constant domain selected from the group consisting of CH and CL. In one aspect of this embodiment, the CL domain is selected from the group consisting of Cxcexa or Cxcex. In another aspect of this embodiment, the two antigen binding sites can each have the same or different antigen binding specificities, thereby providing monospecific or bispecific binding reactivities, respectively. A preferred antigen binding site is a polypeptide comprising an immunoglobulin variable heavy and light chain domain fusion selected from the group consisting of VH/VL and VL/VH, wherein VH is the immunoglobulin variable heavy chain domain and VL is the immunoglobulin variable light chain domain.
In a related embodiment, the invention contemplates a composition comprising a monovalent polypeptide having an amino acid residue sequence according to the formula Vxe2x80x94C, wherein V is an antigen binding site, and C is an immunoglobulin constant domain amino acid residue sequence selected from the group consisting of CH and CL. In one aspect of this embodiment, the CL domain is selected from the group consisting of Cxcexa or Cxcex. In preferred embodiments, the antigen binding site is a polypeptide comprised of an immunoglobulin variable heavy and light chain domain fusion selected from the group consisting of VH/VL and VL/VH.
Also contemplated are polypeptide compositions comprising two or more polypeptides of the present invention operatively linked by disulfide bridges between the immunoglobulin constant domain of each polypeptide, thereby forming multimeric proteins having multiple valencies and antigen-binding specificities. In this embodiment, the immunoglobulin constant region domain is selected from the group consisting of CH and CL wherein the CL domain is selected from the group consisting of Cxcexa or Cxcex, thereby providing a means of sequential purification based upon the different immunological properties of the constant region domains.
Also described are DNA vectors for producing a polypeptide composition of the present invention, and methods of preparing and using the polypeptide compositions.
A. Definitions
Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the xe2x80x9cLxe2x80x9d isomeric form. However, residues in the xe2x80x9cDxe2x80x9d isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature (described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R. 1.822(b)(2)), abbreviations for amino acid residues are shown in the following Table of Correspondence:
It should be noted that all amino acid residue sequences represented herein by formulae have a left-to-right orientation in the conventional direction of amino terminus to carboxy terminus. In addition, the phrase xe2x80x9camino acid residuexe2x80x9d is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those listed in 37 CFR 1.822(b)(4), and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to an amino-terminal group such as NH2 or acetyl or to a carboxy-terminal group such as COOH.
Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1xe2x80x2 carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3xe2x80x2 or 5xe2x80x2 position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a xe2x80x9cbase sequencexe2x80x9d or xe2x80x9cnucleotide sequencexe2x80x9d, and their grammatical equivalents, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5xe2x80x2-terminus to 3xe2x80x2-terminus.
Base Pair (bp): A partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA, uracil (U) is substituted for thymine.
Nucleic Acid: A polymer of nucleotides, either single or double stranded.
Polynucleotide: A polymer of single or double stranded nucleotides. As used herein xe2x80x9cpolynucleotidexe2x80x9d and its grammatical equivalents will include the full range of nucleic acids. A polynucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of two or more deoxyribonucleotides and/or ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art. The polynucleotides of the present invention,include primers, probes, RNA/DNA segments, oligonucleotides or xe2x80x9coligosxe2x80x9d (relatively short polynucleotides), genes, vectors, plasmids, and the like.
Gene: A nucleic acid whose nucleotide sequence codes for an RNA or polypeptide. A gene can be either RNA or DNA.
Duplex DNA: A double-stranded nucleic acid molecule comprising two strands of substantially complementary polynucleotides held together by one or more hydrogen bonds between each of the complementary bases present in a base pair of the duplex. Because the nucleotides that form a base pair can be either a ribonucleotide base or a deoxyribonucleotide base, the phrase xe2x80x9cduplex DNAxe2x80x9d refers to either a DNA-DNA duplex comprising two DNA strands (ds DNA), or an RNA-DNA duplex comprising one DNA and one RNA strand.
Complementary Bases: Nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration.
Complementary Nucleotide Sequence: A sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to that on another single strand to specifically hybridize to it with consequent hydrogen bonding.
Conserved: A nucleotide sequence is conserved with respect to a preselected (reference) sequence if it non-randomly hybridizes to an exact complement of the preselected sequence.
Hybridization: The pairing of substantially complementary nucleotide sequences (strands of nucleic acid) to form a duplex or heteroduplex by the establishment of hydrogen bonds between complementary base pairs. It is a specific, i.e. non-random, interaction between two complementary polynucleotides that can be competitively inhibited.
Nucleotide Analog: A purine or pyrimidine nucleotide that differs structurally from A, T, G, C, or U, but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
DNA Homolog: A nucleic acid having a preselected conserved nucleotide sequence and a sequence coding for a receptor capable of binding a preselected ligand.
Recombinant DNA (rDNA) molecule: A DNA molecule produced by operatively linking two DNA segments. Thus, a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature. rDNA""s not having a common biological origin, i.e., evolutionarily different, are said to be xe2x80x9cheterologousxe2x80x9d.
Vector: A rDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as xe2x80x9cexpression vectorsxe2x80x9d. Particularly important vectors allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.
Receptor: A receptor is a molecule, such as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to another molecule.
Antibody: The term antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules of the compositions of this invention, i.e., molecules that contain an antibody combining site or paratope. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fabxe2x80x2, F(abxe2x80x2)2 and Fv.
Immunoglobulin Constant Region: Immunoglobulin constant regions are those structural portions of an antibody molecule comprising amino acid residue sequences within a given isotype which may contain conservative substitutions therein. Exemplary heavy chain immunoglobulin constant regions are those portions of an immunoglobulin molecule known in the art as CH1, CH2, CH3, CH4 and CH5. An exemplary light chain immunoglobulin constant region is that portion of an immunoglobulin molecule known in the art as CL.
Conservative Substitution: The term conservative substitution as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term conservative substitution also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that molecules having the substituted polypeptide also have the same function.
Antibody Combining Site: An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen. The term immunoreact in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof. Alternatively, an antibody combining site is known as an antigen binding site.
Valency: The term valency refers to the number of potential antigen binding sites in a polypeptide. A polypeptide may be monovalent and contain one antigen binding site or a polypeptide may be bivalent and contain two antigen binding sites. Additionally, a polypeptide may be tetravalent and contain four antigen binding sites. Each antigen binding site specifically binds one antigen. When a polypeptide comprises more than one antigen binding site, each antigen binding site may specifically bind the same or different antigens. Thus, a polypeptide may contain a plurality of antigen binding sites and therefore be multivalent and a polypeptide may specifically bind the same or different antigens.
Specificity: The term specificity refers to the number of potential antigen binding sites which immunoreact with (specifically bind) a given antigen in a polypeptide. The polypeptide may be a single polypeptide or may be two or more polypeptides joined by disulfide bonding. A polypeptide may be monospecific and contain one or more antigen binding sites which specifically bind an antigen or a polypeptide may be bispecific and contain two or more antigen binding sites which specifically bind two immunologically distinct antigens. Thus, a polypeptide may contain a plurality of antigen binding sites which specifically bind the same or different antigens.
Multimeric: A polypeptide comprising more than one polypeptide. A multimer may be dimeric and contain two polypeptides and a multimer may be trimeric and contain three polypeptides. Multimers may be homomeric and contain two or more identical polypeptides or a multimer may be heteromeric and contain two or more nonidentical polypeptides.
Single Chain Antibody: The phrase single chain antibody refers to a single polypeptide comprising one or more antigen binding sites.
Polypeptide: The phrase polypeptide refers to a molecule comprising amino acid residues which do not contain linkages other than amide linkages between adjacent amino acid residues.
Immunologically Distinct: The phrase immunologically distinct refers to the ability to distinguish between two polypeptides on the ability of an antibody to specifically bind one of the polypeptides and not specifically bind the other polypeptide.
Monoclonal Antibody: The phrase monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody.
Fusion Polypeptide: A polypeptide comprised of at least two polypeptides and a linking sequence to operatively link the two polypeptides into one continuous polypeptide. The two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature.
Upstream: In the direction opposite to the direction of DNA transcription, and therefore going from 5xe2x80x2 to 3xe2x80x2 on the non-coding strand, or 3xe2x80x2 to 5xe2x80x2 on the mRNA.
Downstream: Further along a DNA sequence in the direction of sequence transcription or read out, that is traveling in a 3xe2x80x2- to 5xe2x80x2-direction along the non-coding strand of the DNA or 5xe2x80x2- to 3xe2x80x2-direction along the RNA transcript.
Cistron: Sequence of nucleotides in a DNA molecule coding for an amino acid residue sequence and including upstream and downstream DNA expression control elements.
Stop Codon: Any of three codons that do not code for an amino acid, but instead cause termination of protein synthesis. They are UAG, UAA and UGA and are also referred to as a nonsense or termination codon.
Leader Polypeptide: A short length of amino acid sequence at the amino end of a polypeptide, which carries or directs the polypeptide through the inner membrane and so ensures its eventual secretion into the periplasmic space and perhaps beyond. The leader sequence peptide is commonly removed before the polypeptide becomes active.
Reading Frame: Particular sequence of contiguous nucleotide triplets (codons) employed in translation. The reading frame depends on the location of the translation initiation codon.
B. DNA Expression Vectors
A vector of the present invention is a recombinant DNA (rDNA) molecule adapted for receiving and expressing translatable DNA sequences in the form of a polypeptide of this invention. The vector comprises a cassette that includes upstream and downstream translatable DNA sequences operatively linked via a sequence of nucleotides adapted for directional ligation to an insert DNA. The upstream translatable sequence encodes the secretion signal as defined herein. The downstream translatable sequence encodes the that portion of the polypeptide which is expressed when polypeptides of this invention are expressed on the surface of phage. The vector preferably includes DNA expression control sequences for expressing the fusion polypeptide that is produced when an insert translatable DNA sequence (insert DNA) is directionally inserted into the vector via the sequence of nucleotides adapted for directional ligation.
An expression vector is characterized as being capable of expressing, in a compatible host, a structural gene product such as a polypeptide of the present invention.
As used herein, the term xe2x80x9cvectorxe2x80x9d refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked. Preferred vectors are those capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operatively linked.
As used herein with regard to DNA sequences or segments, the phrase xe2x80x9coperatively linkedxe2x80x9d means the sequences or segments have been covalently joined, preferably by conventional phosphodiester bonds, into one strand of DNA, whether in single or double stranded form.
The choice of vector to which a cassette of this invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., vector replication and protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules.
In a preferred embodiment, the vector utilized includes a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, those embodiments that include a prokaryotic replicon also include a gene whose expression confers a selective advantage, such as drug resistance, to a bacterial host transformed therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline. Vectors typically also contain convenient restriction sites for insertion of translatable DNA sequences. Exemplary vectors are the plasmids pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond, Calif.) and pPL and pKK223 available from Pharmacia, (Piscataway, N.J.).
In another preferred embodiment, the vector utilized includes a eukaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in a eukaryotic host cell, such as a mammalian or yeast host cell, transformed therewith. Such replicons are well known in the art and include those replicons which carry the SV40 and 2 xcexc origins of replication for replication in mammalian cells and yeast, respectively. SV40 replicons require the expression of the SV40 large T antigen to support the transient replication of replicons carrying the SV40 origin of replication. In addition, those embodiments that include a eukaryotic replicon also include a gene whose expression confers a selective advantage, such as drug resistance, to a mammalian host transformed therewith. A typical mammalian drug resistance gene is that which confers resistance to G418. Genes whose expression confer a selective advantage to a mammalian host transformed therewith also include those which encode required amino acids, such as leucine, tryptophan, uracil and histidine. Vectors typically also contain convenient restriction sites for insertion of translatable DNA sequences. Exemplary vectors are the plasmids pSVL, pCH110, pMSG, pBPV and pSVK 3 available from Pharmacia (Piscataway, N.J.); the plasmids pBK-CMV, pBK-RSV, pXT1 pSG5, pRS413, pRS414, pRS415, and pRS416 available from Stratagene (La Jolla, Calif.); the plasmids pYES and pYES2 available from Invitrogen (San Diego, Calif.).
In another embodiment, the vector utilized includes an insect replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in an insect host cell, such as an Sf9 host cell, transformed therewith. Such replicons are well known in the art. Vectors typically also contain convenient restriction sites for insertion of translatable DNA sequences. Exemplary vectors are the plasmids pMbac and ppbac and the transfer vector pJVP10Z available from Stratagene.(La Jolla, Calif.).
A sequence of nucleotides adapted for directional ligation, i.e., a polylinker, is a region of the DNA expression vector that (1) operatively links for replication and transport the upstream and downstream translatable DNA sequences and (2) provides a site or means for directional ligation of a DNA sequence into the vector. Typically, a directional polylinker is a sequence of nucleotides that defines two or more restriction endonuclease recognition sequences, or restriction sites. Upon restriction cleavage, the two sites yield cohesive termini to which a translatable DNA sequence can be ligated to the DNA expression vector. Preferably, the two restriction sites provide, upon restriction cleavage, cohesive termini that are non-complementary and thereby permit directional insertion of a translatable DNA sequence into the cassette. In one embodiment, the directional ligation means is provided by nucleotides present in the upstream translatable DNA sequence, downstream translatable DNA sequence, or both. In another embodiment, the sequence of nucleotides adapted for directional ligation comprises a sequence of nucleotides that defines multiple directional cloning means. Where the sequence of nucleotides adapted for directional ligation defines numerous restriction sites, it is referred to as a multiple cloning site.
A translatable DNA sequence is a linear series of nucleotides that provide an uninterrupted series of at least 8 codons that encode a polypeptide in one reading frame.
An upstream translatable DNA sequence encodes a prokaryotic, eukaryotic or insect secretion signal. The secretion signal is a leader peptide domain of protein that targets the protein to the periplasmic membrane of gram negative bacteria, to the endoplasmic reticulum in mammalian cells or to the secretory pathway of the host insect cell.
A preferred secretion signal is a pelB secretion signal in E. coli. The predicted amino acid residue sequences of the secretion signal domain from two pelB gene product variants from Erwinia carotova are shown in Table 1 as described by Lei, et al., Nature, 331:543-546 (1988). A particularly preferred pelB secretion signal is also shown in Table 1.
The leader sequence of the pelB protein has previously been used as a secretion signal for fusion proteins. Better et al., Science, 240:1041-1043 (1988); Sastry et al., Proc. Natl. Acad. Sci. USA, 86:5728-5732 (1989); and Mullinax et al., Proc. Natl. Acad. Sci. USA, 87:8095-8099 (1990).
Amino acid residue sequences for other secretion signal polypeptide domains from E. coli useful in this invention are also listed in Table 1. Oliver, In Neidhard, F. C. (ed.), Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington, D.C., 1:56-69 (1987).
A translatable DNA sequence encoding the pelB secretion signal having the amino acid residue sequence shown in SEQ ID NO 1 is a preferred DNA sequence for inclusion in a DNA expression vector of this invention.
A downstream translatable DNA sequence may encode antigen-binding site(s) and constant region(s) of the polypeptide(s) as described further herein.
Exemplary insect cell secretion signals, melittin and human placental alkaline phosphatase, can be found in the transfer vector pJVP10Z (Stratagene).
A cassette in a DNA expression vector of this invention is the region of the vector that forms, upon insertion of a translatable DNA sequence (insert DNA), a sequence of nucleotides capable of expressing, in an appropriate host, a fusion polypeptide of this invention. The expression-competent sequence of nucleotides is referred to as a cistron. Thus, the cassette comprises DNA expression control elements operatively linked to the upstream and downstream translatable DNA sequences. A cistron is formed when a translatable DNA sequence is directionally inserted (directionally ligated) between the upstream and downstream sequences via the sequence of nucleotides adapted for that purpose. The resulting three translatable DNA sequences, namely the upstream, the inserted and the downstream sequences, are all operatively linked in the same reading frame.
DNA expression control sequences comprise a set of DNA expression signals for expressing a structural gene product and include both 5xe2x80x2 and 3xe2x80x2 elements, as is well known, operatively linked to the cistron such that the cistron is able to express a structural gene product. The 5xe2x80x2 control sequences define a promoter for initiating transcription and a ribosome binding site operatively linked at the 5xe2x80x2 terminus of the upstream translatable DNA sequence.
To achieve high levels of gene expression in E. coli, it is necessary to use not only strong promoters to generate large quantities of mRNA, but also ribosome binding sites to ensure that the mRNA is efficiently translated. In E. coli, the ribosome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotides upstream from the initiation codon (Shine et al., Nature, 254:34 (1975)). The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD) sequence, is complementary to the 3xe2x80x2 end of E. coli 16S mRNA. Binding of the ribosome to mRNA and the sequence at the 3xe2x80x2 end of the mRNA can be affected by several factors:
(i) The degree of complementarity between the SD sequence and 3xe2x80x2 end of the 16S tRNA.
(ii) The spacing and possibly the DNA sequence lying between the SD sequence and the AUG (Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760 (1979a); Roberts et al., Proc. Natl. Acad. Sci. USA, 76:5596 (1979b); Guarente et al., Science, 209:1428 (1980); and Guarente et al., Cell, 20:543 (1980).) Optimization is achieved by measuring the level of expression of genes in plasmids in which this spacing is systematically altered. Comparison of different mRNAs shows that there are statistically preferred sequences from positions xe2x88x9220 to +13 (where the A of the AUG is position 0) (Gold et al., Annu. Rev. Microbiol., 35:365 (1981)). Leader sequences have been shown to influence translation dramatically (Roberts et al., 1979 a, b supra).
(iii) The nucleotide sequence following the AUG, which affects ribosome binding (Taniguchi et al., J. Mol. Biol., 118:533 (1978)).
Useful ribosome binding sites are shown in Table 2 below.
The 3xe2x80x2 control sequences define at least one termination (stop) codon in frame with and operatively linked to the downstream translatable DNA sequence. In addition, eukaryotic vectors contain specific nucleotide sequences which provide the necessary signals for polyadenylation of the transcription product derived from the translatable DNA sequence.
Thus, a DNA expression vector of this invention provides a system for cloning translatable DNA sequences into the cassette portion of the vector to produce a cistron capable of expressing a fusion polypeptide of this invention.
In a preferred embodiment, a DNA expression vector is designed for convenient manipulation in the form of a filamentous phage particle encapsulating a genome according to the teachings of the present invention. In this embodiment, a DNA expression vector further contains a nucleotide sequence that defines a filamentous phage origin of replication such that the vector, upon presentation of the appropriate genetic complementation, can replicate as a filamentous phage in single stranded replicative form and be packaged into filamentous phage particles. This feature provides the ability of the DNA expression vector to be packaged into phage particles for subsequent segregation of the particle, and vector contained therein, away from other particles that comprise a population of phage particles.
A filamentous phage origin of replication is a region of the phage genome, as is well known, that defines sites for initiation of replication, termination of replication and packaging of the replicative form produced by replication. See, for example, Rasched et al., Microbiol. Rev., 50:401-427 (1986); and Horiguchi, J. Mol. Biol., 188:215-223 (1986).
A preferred filamentous phage origin of replication for use in the present invention is a M13, f1 or fd phage origin of replication. A preferred DNA expression vector is the dicistronic expression vector pCOMB8, described in Example 3.
Insofar as a vector of this invention may be manipulated to contain an insert DNA, thereby having the capacity to express a fusion polypeptide, one embodiment contemplates the previously described vectors containing an insert DNA. Particularly preferred vectors containing antibody genes are described in the Examples.
C. Polypeptides and Polypeptide Compositions
In one embodiment, the present invention contemplates a monovalent polypeptide, and compositions thereof, having an amino acid residue sequence according to the formula Vxe2x80x94C, where V is an antigen binding site, and C is an immunoglobulin constant domain amino acid residue sequence selected from the group consisting of CH and CL.
In a preferred embodiment, the antigen binding site is a polypeptide comprising both the heavy and light chain domains of an immunoglobulin molecule, fused into a single polypeptide to provide a ligand binding capability as is well known in the art. It has been discovered that the orientation of the variable domains of the heavy (VH) and light (VL) chains can vary in either the VHxe2x80x94VLxe2x80x94C or VLxe2x80x94VHxe2x80x94C orientation relative to the constant chain domain (C), when read from amino to carboxy terminus. Thus, in a preferred embodiment, the antigen binding site comprises a polypeptide having an immunoglobulin variable heavy and light chain domain selected from the group consisting of VHxe2x80x94VL and VLxe2x80x94VH.
In a preferred embodiment, the immunoglobulin constant domain amino acid residue sequences are those polypeptides which comprise the structural portions of an antibody molecule known in the art as CH1, CH2, CH3 and CH4. A particularly preferred heavy chain constant region is CH1 which lacks the effector functions associated with the CH2 region. These effector functions include the binding of the constant region to the Fc receptors, complement fixation and antibody-depedent cell-mediated cytotoxicity. Also preferred are those polypeptides which are known in the art as CL. Preferrred CL polypeptides are selected from the group consisting of Cxcexa and Cxcex.
The constant and variable regions of a polypeptide can be derived from a variety of sources including mouse, rabbit, rat and human immunoglobulins. Constant and variable regions of a polypeptide which are derived from human immunoglobulins are particularly preferred as they are less immuogenic in humans than polypeptides which are derived from other sources.
As shown herein, the presence of a cysteine residue in the constant domain provides a means for bridging two polypeptides to form a dimeric polypeptide molecule.
Furthermore, insofar as the constant domain can be either a heavy or light chain constant domain (CH or CL, respectively), a. variety of monovalent polypeptide compositions are contemplated by the present invention. For example, light chain constant domains are capable of disulfide bridging to either another light chain constant domain, or to a heavy chain constant domain. In contrast, a heavy chain constant domain can form two independent disulfide bridges, allowing for the possibility of bridging to both another heavy chain and to a light chain, or to form polymers of heavy chains.
Thus, in another embodiment, the invention contemplates a composition comprising a monovalent polypeptide wherein the constant chain domain C has a cysteine residue capable of forming at least one disulfide bridge, and where the composition comprises at least two monovalent polypeptides covalently linked by said disulfide bridge according to the formula: 
In preferred embodiments, the constant chain domain C can be either CL or CH. Where C is CL, the CL polypeptide is preferably selected from the group consisting of Cxcexa and Cxcex. Numerous permutations can be used following the above general formula. Particularly preferred is the formula in which C is CH as follows: 
In a related embodiment, additional monovalent polypeptides having the light chain constant regions, CL, can be associated with the above monovalent polypeptide composition. In this embodiment, the polypeptide composition has a structure according to the formula: 
In another embodiment, the invention contemplates a polypeptide composition comprising a monovalent polypeptide as above except where C is CL having a cysteine residue capable of forming a disulfide bridge, such that the composition contains two monovalent polypeptides covalently linked by said disulfide bridge according to the formula: 
The preparation and use of an exemplary monovalent polypeptide is described further herein, and in the Examples.
In a related embodiment, the present invention describes a bivalent polypeptide, and compositions thereof, where the bivalent polypeptide has an amino acid residue sequence according to the formula Vxe2x80x94Xxe2x80x94V, where V is an antigen binding site as defined above forming a first and second antigen binding site, and X is an amino acid residue sequence of from about 5 to about 120 amino acid residues.
In one embodiment, the first and second antigen binding sites can each have the same binding specificity. In an alternate embodiment, the first and second antigen binding sites have different binding specificities.
Although a variety of polypeptides can be utilized to link the first and second antigen binding sites, it is particularly preferred where X has a means for reversible linkage to a second polypeptide (monovalent or bivalent). A preferred reversible linkage means is a cysteine residue by virtue of the presence of the available sulfur moiety capable of forming a disulfide bridge with other available sulfur moieties.
In addition, it is particularly preferred that X is an immunoglobulin constant domain selected from the group consisting of the heavy chain constant domain (CH) and the light chain constant domain (CL).
Thus, in another embodiment, the invention contemplates a composition comprising a bivalent polypeptide wherein X is the immunoglobulin heavy chain constant domain (CH) having a cysteine residue capable of forming at least one disulfide bridge, and the composition comprises two bivalent polypeptides covalently linked by said disulfide bridge according to the formula: 
In a related embodiment, the invention contemplates a composition comprising a bivalent polypeptide wherein X is a immunoglobulin heavy chain constant domain (CH) having a cysteine residue capable of forming at least one disulfide bridge and an immunoglobulin light chain constant domain (CL) having a cysteine residue capable of forming at least one disulfide bridge, and the composition comprises two bivalent polypeptides covalently linked by said disulfide bridge according to the formula: 
In a related embodiment, additional bivalent polypeptides can be associated with the polypeptide composition wherein the additional bivalent polypeptides have light chain constant regions (CL). The light chain constant regions are selected from the group consisting of Cxcexa and Cxcex. In this embodiment, the polypeptide composition has a structure according to the formula: 
In another embodiment, the invention contemplates a bivalent polypeptide composition defined above in which X is the immunoglobulin light chain constant domain (CL), wherein the light chain constant regions are selected from the group consisting of Cxcexa and Cxcex, having a cysteine residue capable of forming a disulfide bridge, and wherein the composition contains two bivalent polypeptides covalently linked by said disulfide bridge according to the formula: 
The preparation and use of a bivalent polypeptide is described further herein, and in the Examples.
As used herein with regard to polypeptides, the phrase xe2x80x9coperatively linkedxe2x80x9d means that polypeptide fragments, or protein domains represented by polypeptides, have been covalently joined into a single polypeptide polymer, preferably by conventional amide bonds between the adjacent amino acids being linked in the polypeptide.
In one embodiment, the linkage means is the formation of disulfide bonds between two or more polypeptides comprising constant domains resulting in the covalent linkage of two or more polypeptides. In another embodiment, the linkage means is the formation of an amide bond between adjacent amino acid residues resulting in the formation of a polypeptide comprising one or more constant region domains and two or more variable region domains.
In one embodiment, X is an amino acid residue sequence that defines the constant region of an immunoglobulin constant region polypeptide. In a particularly preferred polypeptide X is a heavy chain (CH) or light chain (CL) polypeptide, wherein CL is selected from the group consisting of Cxcexa and Cxcex.
The individual CH and CL domains can be produced in lengths equal to or substantially equal to their naturally occurring lengths. However, in preferred embodiments, the CH and CL polypeptides will generally have fewer than 125 amino acid residues, more usually fewer than about 120 amino acid residues, while normally having greater than 60 amino acid residues, usually greater than about 95 amino acid residues, more usually greater than about 100 amino acid residues. Preferably, the CH will be from about 110 to about 230 amino acid residues in length while CL will be from about 95 to about 214 amino acid residues in length. CH and CL chains sufficiently long to form bispecific and/or bivalent molecules are preferred.
Those individual CH and CL domains which are produced in lengths equal to or substantially equal to their naturally occurring lengths typically contain one or more of the amino acid residue cysteine at or near the 3xe2x80x2 end of the polypeptide. The cysteine residues can form a disulphide bond with cysteine residues present in the same molecule resulting in the formation of one or more intramolecular disulphide bonds or between two or more molecules resulting in the formation of one or more intermolecular disulphide bonds.
In a related embodiment, X is an amino acid residue sequence that defines a portion of the constant region of an immunoglobulin constant region polypeptide. In a particularly preferred polypeptide X is a heavy chain (CH) or light chain (CL) polypeptide, wherein CL is selected from the group consisting of Cxcexa and Cxcex.
The individual CH and CL domains can be produced in lengths lesser than their naturally occurring lengths. In preferred embodiments, the CH and CL polypeptides will generally have fewer than 40 amino acid residues, more usually fewer than about 30 amino acid residues, while normally having greater than 3 amino acid residues, usually greater than about 5 amino acid residues, more usually greater than about 8 amino acid residues. Preferably, the CH will be from about 12 to about 20 amino acid residues in length while CL will be from about 12 to about 25 amino acid residues in length. CH and CL chains sufficiently long to form bispecific and/or bivalent molecules are preferred.
Those individual CH and CL domains which are produced in lengths less than their naturally occurring lengths typically are used to directly link one or more variable regions, V, by the formation of an amide bond between adjacent amino acid residues resulting in the formation of a single polypeptide comprising one or more constant region domains and two or more variable region domains.
In one embodiment, the CH is selected from the group consisting of the immunoglobulin classes IgG, IgA, IgM, IgD and IgE. In a preferred embodiment, the CH is selected from the immunoglobulin class IgG and is selected from the group of IgG1 subclasses consisting of IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, CL is selected from the group consisting of Cxcexa and Cxcex.
Typically the constant region domains of the CH and CL polypeptides will have a lesser variety of sequences than the variable region domains and hence will represent fewer number of unique sequences. Variations in the amino acid residue sequence are represented by the different classes, subclasses, isotypic variants, and allotypes of heavy and light chain immunoglobulins and are well known to those of skill in the art.
In one embodiment, V is an amino acid residue sequence that defines the ligand or antigen binding domain of an immunoglobulin variable region polypeptide. In a particularly preferred polypeptide V is a VH or VL polypeptide.
A polypeptide of the present invention assumes a conformation having a binding site specific for, as evidenced by its ability to be competitively inhibited, a preselected or predetermined ligand such as an antigen, hapten, enzymatic substrate and the like. In one embodiment, a receptor of this invention is a ligand binding polypeptide that forms an antigen binding site which specifically binds to a preselected antigen to form a complex having a sufficiently strong binding between the antigen and the binding site for the complex to be isolated. When the receptor is an antigen binding polypeptide its affinity or avidity is generally greater than 105 Mxe2x88x921 more usually greater than 106 and preferably greater than 108 Mxe2x88x921.
One or more of the different polypeptide chains is preferably derived from the variable region of the light and heavy chains of an immunoglobulin. Typically, polypeptides comprising the light (VL) and heavy (VH) variable regions are employed together for binding the preselected ligand.
The individual VH and VL domains can be produced in lengths equal to or substantially equal to their naturally occurring lengths. However, in preferred embodiments, the VH and VL polypeptides will generally have fewer than 125 amino acid residues, more usually fewer than about 120 amino acid residues, while normally having greater than 60 amino acid residues, usually greater than about 95 amino acid residues, more usually greater than about 100 amino acid residues. Preferably, the VH will be from about 110 to about 230 amino acid residues in length while VL will be from about 95 to about 214 amino acid residues in length. VH and VL chains sufficiently long to form single chain polypeptides or Fabs are preferred.
Typically the variable regions of the VH and VL polypeptides will have a greater variety of sequences than the constant regions and, based on the present strategy, the variable regions can be further modified to permit a variation of the normally occurring VH and VL chains. A synthetic polynucleotide can be employed to vary one or more amino acid in a variable region to alter the binding affinity or binding specificity of a variable region.
D. Methods for Producing a Monovalent or Bivalent Polypeptide
1. General Rationale
In one embodiment the present invention provides a method for producing novel polypeptides as described further herein. Although the invention describes both monovalent and bivalent polypeptides, their general preparation depends upon the construction of one or more DNA expression vectors in which a gene encoding an antigen binding site (V) is operatively linked in-frame to a gene encoding an immunoglobulin constant domain (C) so as to form a fusion protein comprised of polypeptide domains not normally associated in nature.
In the preparation of a monovalent polypeptide, having an amino acid residue sequence defined herein by the formula Vxe2x80x94C, the method involves operatively linking first and second nucleotide sequences that code for V and C amino acid residue sequences, respectively, to a nucleotide expression vector capable of expressing the monovalent polypeptide. The V and C region genes are linked in frame so as to preserve the frame of the expressed polypeptide, as is well known.
The antibody binding site domain (V) is comprised of heavy and light chain variable domain amino acid residue sequences, and therefore, nucleotide sequences coding the V region can be ordered on the V region gene in either the VHxe2x80x94VL or VLxe2x80x94VH orientation as described further herein.
The immunoglobulin constant domain (C) can be a CH or CL domain, and therefore, nucleotide sequences encoding the C region can be comprised of either CH or CL genes. The nucleotide sequences encoding the CL region can be comprised of either Cxcexa or Cxcex genes. The C region gene is operatively linked to the V region genes so as to be in the same reading frame with the encoded V and C domain amino acid residue sequences.
In the preparation of a bivalent polypeptide, having an amino acid residue sequence defined herein by the formula Vxe2x80x94Xxe2x80x94V, the method involves operatively linking first, second and third nucleotide sequences that code for a first V domain, an X domain and a second V domain amino acid residue sequences, respectively, to a nucleotide expression vector capable of expressing the bivalent polypeptide. The three domain genes (V, X and V) are linked in the same reading frame so as to preserve the frame of the expressed polypeptide.
In one embodiment, the DNA expression vector can be used to express one or more different polypeptides of this invention, and by virtue of the presence of the cysteine residues in the constant domain, the expressed polypeptides can associate to form dimeric and multimeric proteins, thereby forming polypeptide compositions with multiple polypeptide subunits and potentially multiple antigen binding specificities.
In preferred embodiments using a monovalent polypeptide, the disclosure provides for the combined expression of two or more monovalent polypeptides, each having a different antigen-binding site specificity, so as to form upon disulfide bridging a dimeric or multimeric protein composition having multiple ligand binding specificities.
A particular aspect of this embodiment is the production of dimeric polypeptide compositions in which one monovalent polypeptide contains a heavy chain constant domain, and the other monovalent polypeptide contains a light chain constant domain, thereby utilizing the natural disulfide bridging mechanism between heavy and light chains normally found in immunoglobulins to approximate a heavy chain-light chain heterodimer. To that end, the method contemplates the preparation of a first monovalent polypeptide in which a first antibody binding site is fused to CH1 and expressed in a heavy chain cloning cassette, and a second monovalent polypeptide in which a second antibody binding site is fused to a kappa constant domain (Cxcexa) in a light chain cloning cassette. Two independent libraries of the first and second directed molecules are thereby constructed and recombined randomly to generate combinatorial bispecific antigen-binding polypeptide molecules. Fusion of the CH1 and kappa constant domain results in predominantly heterodimer formation which could be readily purified on a protein G column by FPLC.
In another particular aspect, the first antibody binding site is fused to a heavy chain constant domain (CH) and expressed in a heavy chain cloning cassette, and the second antibody binding site is fused to a lambda constant domain (Cxcex) in a light chain cloning cassette. Two independent libraries of the first and second molecules are thereby constructed and recombined randomly to generate combinatorial bispecific antigen-binding polypeptide molecules. Fusion of the CH and Cxcex results in predominantly heterodimer formation which can be readily purified on a protein G column by FPLC.
In another particular aspect of this embodiment is the production of dimeric polypeptide compositions in which both monovalent polypeptides contain a light chain constant domain wherein the light chain constant domain is Cxcexa, thereby utilizing the disulfide bridging mechanism between the light chains to approximate a light chain-light chain heterodimer. To that end, the method contemplates the preparation of first and second monovalent polypeptides in which the first and second antibody binding sites are fused to a kappa constant domain (Cxcexa) and expressed in the first and second light chain cloning cassettes. Two independent libraries of the first and second molecules are thereby constructed and recombined randomly to generate combinatorial bispecific antigen-binding polypeptide molecules. Fusion of the two kappa constant domains results in predominantly heterodimer formation which could be readily purified on a column which binds the kappa light chain.
In an alternate aspect of this embodiment is the production of dimeric polypeptide compositions in which both monovalent polypeptides contain a light chain constant domain wherein the light chain constant domains are selected from the group consisting of Cxcexa and Cxcex, thereby utilizing the disulfide bridging mechanism between the light chains to approximate a light chain-light chain heterodimer. To that end, the method contemplates the preparation of a first monovalent polypeptide in which a first antibody binding site is fused to a kappa constant domain (Cxcexa) and expressed in a light chain cloning cassette, and a second monovalent polypeptide in which a second antibody binding site is fused to a lambda constant domain (Cxcex) in a light chain cloning cassette. Two independent libraries of the first and second directed molecules are thereby constructed and recombined randomly to generate combinatorial bispecific antigen-binding polypeptide molecules. Fusion of the kappa and lambda constant domains results in predominantly heterodimer formation which could be readily purified by sequential purification with a first column which binds the kappa light chain and a second column which binds the lambda light chain resulting in a relatively homogenous product containing the kappa and lambda constant domains. Also contemplated is a first column which binds the lambda light chain and a second column which binds the kappa light chain resulting in a relatively homogenous product containing the kappa and lambda constant domains.
In an additional aspect of this embodiment is the production of dimeric polypeptide compositions in which both monovalent polypeptides contain a light chain constant domain wherein the light chain constant domain is Cxcex, thereby utilizing the disulfide bridging mechanism between the light chains to approximate a light chain-light chain heterodimer. To that end, the method contemplates the preparation of first and second monovalent polypeptides in which the first and second antibody binding sites are fused to separate lambda constant domains (Cxcex) and expressed in first and second light chain cloning cassettes. Two independent libraries of the first and second molecules are thereby constructed and recombined randomly to generate combinatorial bispecific antigen-binding polypeptide molecules. Fusion of the lambda constant domains results in predominantly heterodimer formation which could be readily purified by a column which binds the lambda light chain.
In another embodiment, a bispecific tetravalent polypeptide molecule can be prepared. Following the construction of cloning vectors for expressing whole antibodies in mammalian cells with readily compatible cloning sites, one transfers the antibody binding site domain (V in the form of VHxe2x80x94VL) directly to the appropriate cloning cassette generating first an entire heavy chain and second a light chain-like polypeptide combination. upon expression, the molecule produced from such a construction assembles into a first Hxe2x80x94L like heterodimer and then self associates to form a molecule that is tetravalent (four binding sites) yet bispecific for the desired specificities. This could be a very potent molecule in a therapeutic setting since this would permit the utilization of the natural effector of the antibody Fc region.
Extending the possible combinations further, one can prepare trispecific or trivalent or hexavalent polypeptide molecules. To that end, a bivalent polypeptide vector is first prepared having a heavy chain constant domain. A monovalent polypeptide vector is also prepared having a light chain constant domain, and the two polypeptides are co-expressed in the same expression medium. The light chain constant domains are selected from the group consisting of kappa (Cxcexa) and lambda (Cxcex) light chain constant domains. The expressed polypeptides assemble first to form a monovalent-bivalent polypeptide pair, thereby forming a trivalent polypeptide composition, and the formed polypeptide self-associates to produce a hexavalent polypeptide.
In a related embodiment, one can construct a tetraspecific and tetravalent polypeptide composition by using two bivalent polypeptides in which each antibody binding site is directed to a different ligand. The two bivalent polypeptide chains are co-expressed in a single expression medium, allowing their assembly to form a dimeric polypeptide composition that is tetravalent and tetraspecific.
2. Production of Immunoglobulin Variable and Constant Domain Genes
Methods for preparing fragments of genomic DNA from which immunoglobulin variable and constant region genes can be cloned are well known in the art. See for example Herrmann et al., Methods In Enzymol., 152:180-183, (1987); Frischauf, Methods In Enzymol., 152:183-190 (1987); Frischauf, Methods In Enzymol., 152:190-199 (1987); and DiLella et al., Methods In Enzymol., 152:199-212 (1987). (The teachings of the references cited herein are hereby incorporated by reference.)
The desired immunoglobulin genes can be isolated from either genomic material containing the gene expressing the variable region or the messenger RNA (mRNA) which represents a transcript of the variable region. The difficulty in using the genomic DNA from other than non-rearranged B lymphocytes is in juxtaposing the sequences coding for the variable region, where the sequences are separated by introns. The DNA fragment(s) containing the proper exons must be isolated, the introns excised, and the exons then spliced in the proper order and in the proper orientation. For the most part, this will be difficult, so that the alternative technique employing rearranged B cells will be the method of choice because the V, D and J immunoglobulin gene regions have translocated to become adjacent, so that the sequence is continuous (free of introns) for the entire variable regions.
Where mRNA is utilized the cells will be lysed under RNase inhibiting conditions. In one embodiment, the first step is to isolate the total cellular mRNA. Poly A+ mRNA can then be selected by hybridization to an oligo-dT cellulose column. Selected polyA+ mRNA is then converted to double-stranded DNA and inserted into an appropriate vector by methods well known to those of skill in the art to produce a cDNA library. The cDNA library contains DNA inserts representing mRNAs expressed in the cells, including those which code for the expression of heavy and light chain polypeptides, from which the mRNA was derived.
The presence of DNA inserts coding for the heavy and/or light chain polypeptides present in the cDNA library can be identified by hybridization with single-stranded DNA of the appropriate genes. Conveniently, the sequences coding for the constant portion of the heavy and/or light chain polypeptides can be used as polynucleotide probes, which sequences can be obtained from available sources. See for example, Early and Hood, Genetic Engineering, Setlow and Hollaender, eds., Vol. 3, Plenum Publishing Corporation, NY, (1981), pages 157-188; and Kabat et al., Sequences of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987).
In preferred embodiments, the preparation containing the total cellular mRNA is first enriched for the presence of CH, CL, VH and/or VL coding mRNA. A preferred source of mRNA is a hybridoma capable of secreting monoclonal antibodies directed to a specific antigen. An additional preferred source of mRNA are-cells which express antibodies directed to a plurality of antigens isolated from mouse, rat, rabbit and human. A particularly preferred source of cells which express antibody are isolated from human. Enrichment is typically accomplished by subjecting the total mRNA preparation or partially purified mRNA product thereof to a primer extension reaction employing a polynucleotide synthesis primer as described herein. Exemplary methods for producing VH and VL gene repertoires using polynucleotide synthesis primers are described in PCT Application No. PCT/US 90/02836 (International Publication No. WO 90/14430). Particularly preferred methods for producing a gene repertoire rely on the use of preselected oligonucleotides as primers in a polymerase chain reaction (PCR) to form PCR reaction products as described herein.
In preferred embodiments, isolated B cells are immunized in vitro against a preselected antigen. In vitro immunization is defined as the clonal expansion of epitope-specific B cells in culture, in response to antigen stimulation. The end result is to increase the frequency of antigen-specific B cells in the immunoglobulin repertoire, and thereby decrease the number of clones in an expression library that must be screened to identify a clone expressing an antibody of the desired specificity. The advantage of in vitro immunization is that human monoclonal antibodies can be generated against a limitless number of therapeutically valuable antigens, including toxic or weak immunogens. For example, antibodies specific for the polymorphic determinants of tumor-associated antigens, rheumatoid factors, and histocompatibility antigens can be produced, which can not be elicited in immunized animals. In addition, it may be possible to generate immune responses which are normally suppressed in vivo. Exemplary immune responses which are normally suppressed in vivo are those responses to human cell surface markers.
In vitro immunization can be used to give rise to either a primary or secondary immune response. A primary immune response, resulting from first time exposure of a B cell to an antigen, results in clonal expansion of epitope-specific cells and the secretion of IgM antibodies with low to moderate apparent affinity constants (106-108 Mxe2x88x921). Primary immunization of human splenic and tonsillar lymphocytes in culture can be used to produce monoclonal antibodies against a variety of antigens, including cells, peptides, macromolecule, haptens, and tumor-associated antigens. Memory B cells from immunized donors can also be stimulated in culture to give rise to a secondary immune response characterized by clonal expansion and the production of high affinity antibodies ( greater than 109 Mxe2x88x921) of the IgG isotype, particularly against viral antigens by clonally expanding sensitized lymphocytes derived from seropositive individuals.
In one embodiment, peripheral blood lymphocytes are depleted of various cytolytic cells that appear to down-modulate antigen-specific B cell activation. When lysosome-rich subpopulations (natural killer cells, cytotoxic and suppressor T cells, monocytes) are first removed by treatment with the lysosmotropic methyl ester of leucine, the remaining cells (including B cells, T helper cells, accessory cells) respond antigen-specifically during in vitro immunization. The lymphokine requirements for inducing antibody production in culture are satisfied by a culture supernatant from activated, irradiated T cells.
In addition to in vitro immunization, cell panning (immunoaffinity absorption) can be used to further increase the frequency of antigen-specific B cells. Techniques for selecting B cell subpopulations via solid-phase antigen binding are well established. Panning conditions can be optimized to selectively enrich for B cells which bind with high affinity to a variety of antigens, including cell surface proteins. Panning can be used alone, or in combination with in vitro immunization to increase the frequency of antigen-specific cells above the levels which can be obtained with either technique alone. Immunoglobulin expression libraries constructed from enriched populations of B cells are biased in favor of antigen-specific antibody clones, and thus, enabling identification of clones with the desired specificities from smaller, less complex libraries.
3. Preparation of Polynucleotide Primers
The term xe2x80x9cpolynucleotidexe2x80x9d as used herein in reference to primers, probes and nucleic acid fragments or segments to be synthesized by primer extension is defined as a molecule comprised of two or more deoxyribonucleotide or ribonucleotides, preferably more than 3. Its exact size will depend on many factors, which in turn depends on the ultimate conditions of use.
The term xe2x80x9cprimerxe2x80x9d as used herein refers to a polynucleotide whether purified from a nucleic acid restriction digest or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, reverse transcriptase and the like, and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency, but may alternatively be in double stranded form. If double stranded, the primer is first treated to separate it from its complementary strand before being used to prepare extension products. Preferably, the primer is a polydeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agents for polymerization. The exact lengths of the primers will depend on may factors, including temperature and the source of primer. For example, depending on the complexity of the target sequence, a polynucleotide primer typically contains 15 to 25 or more nucleotides, although it can contain fewer nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with template.
The primers used herein are selected to be xe2x80x9csubstantiallyxe2x80x9d complementary to the different strands of each specific sequence to be synthesized or amplified. This means that the primer must be sufficiently complementary to non-randomly hybridize with its respective template strand. Therefore, the primer sequence may or may not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5xe2x80x2 end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Such non-complementary fragments typically code for an endonuclease restriction site. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided the primer sequence has sufficient complementarily with the sequence of the strand to be synthesized or amplified to non-randomly hybridize therewith and thereby form an extension product under polynucleotide synthesizing conditions.
Primers of the present invention may also contain a DNA-dependent RNA polymerase promoter sequence or its complement. See for example, Krieg et al., Nucl. Acids Res., 12:7057-70 (1984); Studier et al., J. Mol. Biol., 189:113-130 (1986); and Molecular Cloning: A Laboratory Manual. Second Edition, Maniatis et al., eds., Cold Spring Harbor, N.Y. (1989).
When a primer containing a DNA-dependent RNA polymerase promoter is used the primer is hybridized to the polynucleotide strand to be amplified and the second polynucleotide strand of the DNA-dependent RNA polymerase promoter is completed using an inducing agent such as E. coli DNA polymerase I, or the Klenow fragment of E. coli DNA polymerase. The starting polynucleotide is amplified by alternating between the production of an RNA polynucleotide and DNA polynucleotide.
Primers may also contain a template sequence or replication initiation site for a RNA-directed RNA polymerase. Typical RNA-directed RNA polymerase include the QB replicase described by Lizardi et al., Biotechnology, 6:1197-1202 (1988). RNA-directed polymerases produce large numbers of RNA strands from a small number of template RNA strands that contain a template sequence or replication initiation site. These polymerases typically give a one million-fold amplification of the template strand as has been described by Kramer et al., J. Mol. Biol., 89:719-736 (1974).
The polynucleotide primers can be prepared using any suitable method, such as, for example, the phosphotriester or phosphodiester methods see Narang et al., Meth. Enzymol., 68:90, (1979); U.S. Pat. No. 4,356,270; and Brown et al., Meth. Enzymol., 68:109, (1979).
The choice of a primer""s nucleotide sequence depends on factors such as the distance on the nucleic acid from the region coding for the desired receptor, its hybridization site on the nucleic acid relative to any second primer to be used, the number of genes in the repertoire it is to hybridize to, and the like. Additional nucleotide sequences may also be added to the 5xe2x80x2 non-priming portion of the primer. Such nucleotide sequences may represent restriction enzyme recognition sites, translational stop codons and the like.
4. Polymerase Chain Reaction to Produce Cloned Variable and Constant Immunoglobulin Genes
The strategy used for cloning the CH, CL, VH and/or VL genes contained within a repertoire will depend, as is well known in the art, on the type, complexity, and purity of the nucleic acids making up the repertoire. Other factors include whether or not the genes are contained in one or a plurality of repertoires and whether or not they are to be amplified and/or mutagenized.
The immunoglobulin gene repertoires are comprised of polynucleotide coding strands, such as mRNA and/or the sense strand of genomic DNA. If the repertoire is in the form of double stranded genomic DNA, it is usually first denatured, typically by melting, into single strands. A repertoire is subjected to a PCR reaction by treating (contacting) the repertoire with a PCR primer pair, each member of the pair having a preselected nucleotide sequence. The PCR primer pair is capable of initiating primer extension reactions by hybridizing to nucleotide sequences, preferably at least about 10 nucleotides in length and more preferably at least about 20 nucleotides in length, conserved within the repertoire. The first primer of a PCR primer pair is sometimes referred to herein as the xe2x80x9csense primerxe2x80x9d because it hybridizes to the coding or sense strand of a nucleic acid. In addition, the second primer of a PCR primer pair is sometimes referred to herein as the xe2x80x9canti-sense primerxe2x80x9d because it hybridizes to a non-coding or anti-sense strand of a nucleic acid, i.e., a strand complementary to a coding strand.
The PCR reaction is performed by mixing the PCR primer pair, preferably a predetermined amount thereof, with the nucleic acids of the repertoire, preferably a predetermined amount thereof, in a PCR buffer to form a PCR reaction admixture. The admixture is maintained under polynucleotide synthesizing conditions for a time period, which is typically predetermined, sufficient for the formation of a PCR reaction product, thereby producing a plurality of different VH-coding and/or VL-coding DNA homologs.
A plurality of first primer and/or a plurality of second primers can be used in each amplification, e.g., one species of first primer can be paired with a number of different second primers to form several different primer pairs. Alternatively, an individual pair of first and second primers can be used. In any case, the amplification products of amplifications using the same or different combinations of first and second primers can be combined to increase the diversity of the gene library.
In another strategy, the object is to clone the immunoglobulin genes from a repertoire by providing a polynucleotide complement of the repertoire, such as the anti-sense strand of genomic dsDNA or the polynucleotide produced by subjecting mRNA to a reverse transcriptase reaction. Methods for producing such complements are well known in the art.
The PCR reaction is performed using any suitable method. Generally it occurs in a buffered aqueous solution, i.e., a PCR buffer, preferably at a pH of 7-9, most preferably about 8. Preferably, a molar excess (for genomic nucleic acid, usually about 106:1 primer:template) of the primer is admixed to the buffer containing the template strand. A large molar excess is preferred to improve the efficiency of the process.
The PCR buffer also contains the deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP and a polymerase, typically thermostable, all in adequate amounts for primer extension (polynucleotide synthesis) reaction. The resulting solution (PCR admixture) is heated to about 90xc2x0 C.-100xc2x0 C. for about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period the solution is allowed to cool to 54xc2x0 C., which is preferable for primer hybridization. The synthesis reaction may occur at from room temperature up to a temperature above which the polymerase (inducing agent) no longer functions efficiently. Thus, for example, if DNA polymerase is used as inducing agent, the temperature is generally no greater than about 40xc2x0 C. An exemplary PCR buffer comprises the following: 50 mM KCl; 10 mM Tris-HCl; pH 8.3; 1.5 mM MgCl2; 0.001% (wt/vol) gelatin, 200 uM dATP; 200 uM dTTP; 200 uM dCTP; 200 uM dGTP; and 2.5 units Thermus aquaticus DNA polymerase I (U.S. Pat. No. 4,889,818) per 100 microliters of buffer.
The inducing agent may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptase, and other enzymes, including heat-stable enzymes, which will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each nucleic acid strand. Generally, the synthesis will be initiated at the 3xe2x80x2 end of each primer and proceed in the 5xe2x80x2 direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be inducing agents, however, which initiate synthesis at the 5xe2x80x2 end and proceed in the above direction, using the same process as described above.
The inducing agent also may be a compound or system which will function to accomplish the synthesis of RNA primer extension products, including enzymes. In preferred embodiments, the inducing agent may be a DNA-dependent RNA polymerase such as T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. These polymerases produce a complementary RNA polynucleotide. The high turn over rate of the RNA polymerase amplifies the starting polynucleotide as has been described by Chamberlin et al., The Enzymes, ed. P. Boyer, PP. 87-108, Academic Press, New York (1982). Another advantage of T7 RNA polymerase is that mutations can be introduced into the polynucleotide synthesis by replacing a portion of cDNA with one or more mutagenic oligodeoxynucleotides (polynucleotides) and transcribing the partially-mismatched template directly as has been previously described by Joyce et al., Nuc. Acid Res., 17:711-722 (1989). Amplification systems based on transcription have been described by Gingeras et al., in PCR Protocols, A Guide to Methods and Applications, pp 245-252, Academic Press, Inc., San Diego, Calif. (1990).
If the inducing agent is a DNA-dependent RNA polymerase and therefore incorporates ribonucleotide triphosphates, sufficient amounts of ATP, CTP, GTP and UTP are admixed to the primer extension reaction admixture and the resulting solution is treated as described above.
The newly synthesized strand and its complementary nucleic acid strand form a double-stranded molecule which can be used in the succeeding steps of the process.
The first and/or second PCR reactions discussed above can advantageously be used to incorporate into the receptor a preselected epitope useful in immunologically detecting and/or isolating a receptor. This is accomplished by utilizing a first and/or second polynucleotide synthesis primer or expression vector to incorporate a predetermined amino acid residue sequence into the amino acid residue sequence of the receptor.
After producing immunoglobulin gene DNA homologs for a plurality of different immunoglobulin genes within the repertoires, the DNA molecules are typically further amplified. While the DNA molecules can be amplified by classic techniques such as incorporation into an autonomously replicating vector, it is preferred to first amplify the molecules by subjecting them to a polymerase chain reaction (PCR) prior to inserting them into a vector. PCR is typically carried out by thermocycling i.e., repeatedly increasing and decreasing the temperature of a PCR reaction admixture within a temperature range whose lower limit is about 10xc2x0 C. to about 40xc2x0 C. and whose upper limit is about 90xc2x0 C. to about 100xc2x0 C. The increasing and decreasing can be continuous, but is preferably phasic with time periods of relative temperature stability at each of temperatures favoring polynucleotide synthesis, denaturation and hybridization.
PCR amplification methods are described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188, and at least in several texts including xe2x80x9cPCR Technology: Principles and Applications for DNA Amplificationxe2x80x9d, H. Erlich, ed., Stockton Press, New York (1989); and xe2x80x9cPCR Protocols: A Guide to Methods and Applicationsxe2x80x9d, Innis et al., eds., Academic Press, San Diego, Calif. (1990).
In preferred embodiments only one pair of first and second primers is used per amplification reaction. The amplification reaction products obtained from a plurality of different amplifications, each using a plurality of different primer pairs, are then combined.
However, the present invention also contemplates DNA homolog production via co-amplification (using two pairs of primers), and multiplex amplification (using up to about 8, 9 or 10 primer pairs).
In preferred embodiments, the PCR process is used not only to produce a library of DNA molecules, but also to induce mutations within the library or to create diversity from a single parental clone and thereby provide a library having a greater heterogeneity. First, it should be noted that the PCR process itself is inherently mutagenic due to a variety of factors well known in the art. Second, in addition to the mutation inducing variations described in the above referenced U.S. Pat. No. 4,683,195, other mutation inducing PCR variations can be employed. For example, the PCR reaction admixture, can be formed with different amounts of one or more of the nucleotides to be incorporated into the extension product. Under such conditions, the PCR reaction proceeds to produce nucleotide substitutions within the extension product as a result of the scarcity of a particular base. Similarly, approximately equal molar amounts of the nucleotides can be incorporated into the initial PCR reaction admixture in an amount to efficiently perform X number of cycles, and then cycling the admixture through a number of cycles in excess of X, such as, for instance, 2X. Alternatively, mutations can be induced during the PCR reaction by incorporating into the reaction admixture nucleotide derivatives such as inosine, not normally found in the nucleic acids of the repertoire being amplified. During subsequent in vivo DNA synthesis and replication of the nucleic acids in a host cell, the nucleotide derivative will be replaced with a substitute nucleotide thereby inducing a point mutation.
5. Ligation Reactions to Produce Vectors
In preparing a vector of this invention, a ligation admixture is prepared as described above, and the admixture is subjected to ligation conditions for a time period sufficient for the admixed polypeptide genes to ligate (become operatively linked) to the plurality of DNA expression vectors to form the library.
Ligation conditions are conditions selected to favor a ligation reaction wherein a phosphodiester bond is formed between adjacent 3xe2x80x2 hydroxyl and 5xe2x80x2 phosporyl termini of DNA. The ligation reaction is preferably catalyzed by the enzyme T4 DNA ligase. Ligation conditions can vary in time, temperature, concentration of buffers, quantities of DNA molecules to be ligated, and amounts of ligase, as is well known. Preferred ligation conditions involve maintaining the ligation admixture at 4 degrees Centigrade (4xc2x0 C.) to 12xc2x0 C. for 1 to 24 hours in the presence of 1 to 10 units of T4 DNA ligase per milliliter (ml) and about 1 to 2 micrograms (ug) of DNA. Ligation buffer in a ligation admixture typically contains 0.5 M Tris-HCl (pH 7.4), 0.01 M MgCl2, 0.01 M dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg/ml bovine serum albumin (BSA). Other ligation buffers can also be used.
Exemplary ligation reactions are described in Example 3.
6. Purification of Polypeptides
Conventional methods of purifying polypeptides comprising antibodies include precipitation and column chromatography and are well known to one of skill in the purification arts. The method of purification used is dependent upon several factors including the purity required, the source of the antibody, the intended use for the antibody, the species in which the antibody was produced, the class of the antibody and, when the antibody is a monoclonal antibody, the subclass of the antibody.
A commonly used method of purification is affinity chromatography in which the antibody to be purified is bound by protein A, protein G or by an anti-immunoglobulin antibody. Another method of affinity chromatography, which is well known to those of skill in the art, is the specific binding of the antibody to its respective antigen.
a) Purifiction with Protein A or G
In one embodiment, an antibody is purified in a single purification step by specific binding of the antibody to protein A or G using well known methods. Non-specifically bound molecules are removed in a wash step and the specifically bound antibody is eluted.
b) Purification with Antigen
In another embodiment, an antigen is effective in the isolation of polypeptides comprising a variable region domain which specifically binds the antigen.
In an alternative embodiment, the antibody comprising a variable region domain is specifically bound to a single antigen. In a sequential purification procedure, the bispecific antibody comprising two or more variable domains is specifically bound to a first antigen and then to a second antigen. In a preferred embodiment, the first and second antigen are selected from the group consisting of progesterone, NPN, DPN, CD3 and CD4.
In an alternative embodiment, a bispecific antibody comprising two or more variable regions is purified by sequential purification by specifically binding the antibody to a first antigen in a first purification step and to a second antigen in a second purification step.
c) Purification with Anti-IgG Antibodies
Variations in the amino acid residue sequence of the constant region domains of antibodies result in constant region domains which are immunologically distinct. Thus, antibodies can be produced which specifically bind specific classes, subclasses, isotypic variants, and even allotypes of the heavy and light chain constant regions. Such antibodies which specifically bind to the immunologically distinct constant region domains can be used to distinguish between and to purify polypeptides comprising the constant region domains.
In one embodiment, an anti-immunoglobulin antibody is effective in the isolation of polypeptides comprising a constant region domain which is immunologically distinct from other constant region domains.
The method of purifying an antibody with an anti-immunoglobulin antibody can be either a single purification procedure or a sequential purification procedure. Methods of single and sequential purification are well known to those in the purification arts. In a single-step purification procedure, the antibody is specifically bound by a single anti-immunoglobulin antibody. Non-specifically bound molecules are removed in a wash step and the specifically bound molecules are specifically eluted. In a sequential purification procedure, the antibody is specifically bound to a first anti-immunoglobulin antibody, non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted. The eluant from the first anti-immunoglobulin antibody is then specifically bound to a second anti-immunoglobulin antibody. The non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted. In a preferred embodiment, the antibody is sequentially purified by a first and second anti-immunoglobulin antibody selected from the group consisting of antibodies which specifically bind heavy and light chain constant regions. In a more preferred embodiment, the antibody is sequentially purified by a first and second anti-immunoglobulin antibody selected from the group consisting of antibodies which specifically bind the heavy chain constant region of IgG and light chain constant regions of kappa and lambda. In an even more preferred embodiment, the anti-immunoglobulin antibody is selected from the group consisting of antibodies which specifically bind the light chain constant regions of kappa and lambda.
In a preferred embodiment, an antibody of this invention comprising the kappa and lambda constant region domains is sequentially purified by affinity chromatography. In this method, first and second affinity columns are prepared comprising anti-kappa and anti-lambda antibodies. The anti-kappa and anti-lambda antibodies used correspond to the genus of polypeptide to be purified, e.g., if a human kappa constant region is to be purified, an anti-human kappa light chain antibody is used. First, 16 mgs of goat anti-human kappa light chain antibody is admixed with 8 ml Gammabind beads (Pharmacia, Piscataway, N.J.). The beads and antibody are mixed on a rocker at room temperature for 1 hour. The beads are washed twice with 10 volumes of 0.2 M sodium borate, pH 9.0, to remove unbound antibody. The washed beads are resuspended in 10 volumes of 0.2 M sodium borate, pH 9.0. The concentration of (dimethylaminomethyl)phenol (DMP) is brought to 20 mM. Ten microliters of the beads are removed to determine coupling efficiency by gel electrophoresis. The beads are admixed with the DMP on a rocker at room temperature for 30 minutes. Ten microliters of the beads are removed to determine coupling efficiency by gel electrophoresis. The coupling reaction is stopped by washing the beads once in 0.2 M ethanolamine, pH 8.0, and subsequently incubating at room temperature in 0.2 M ethanolamine, pH 8.0, on a rocker. The beads are allowed to settle or are centrifuged and the beads resuspended in PBS and thimerosol (0.05% w/v) added prior to storage. Prior to the purification of antibodies of this invention, the column is washed with 3 column volumes of 10.8% buffer B (buffer B is 0.1 M sodium phosphate (dibasic) and 0.5 M NaCl) and 98.2% buffer A (buffer A is 0.05 M citric acid (free acid) and 0.5 M NaCl) to remove non-DMP bound antibodies. The efficiency of the coupling reaction is determined by comparison of the two aliquots of beads which were removed before and after the coupling reaction by SDS PAGE gel electrophoresis. Beads with coupled antibody are used to prepare a column.
The pH of a solution containing antibodies to be purified is adjusted to 7.4. Supernatants containing antibodies are filtered through a 0.22 micron filter. Flow rates for loading samples containing antibody are generally 1 to 2 ml per minute. Washing and elution flow rates are usually. 3 to 4 ml per minute. The column is equilibrated with at least 3 column volumes of 87.2% buffer B.
Typically, 20 ml of sample containing the antibody to be purified is loaded onto a 10 ml column of beads with coupled anti-kappa light chain antibody. The sample is then washed with 100 ml of 87.2% buffer B and 12.8% buffer A. The bound antibody is then eluted with 10.8% buffer B and 98.2% buffer A which is pH 2.3. The eluted antibody is neutralized with 1 M tris, pH 9.0. Eluted fractions containing antibody are concentrated until the desired antibody concentration is reached. The antigen binding activity of the purified antibody can be determined in an ELISA assay as described in the Examples and the purity of the antibody assessed by SDS PAGE gel analysis.
Affinity matrices comprising anti-immunoglobulin antibodies can be prepared using the methods described above with anti-lambda and anti-heavy chain constant domains antibodies to purify antibodies comprising lambda and heavy chain constant domains, respectively.
In an alternative embodiment, a bispecific antibody is purified by sequential purification by specifically binding the antibody to a first antigen in a first purification step and to a second antigen in a second purification step by methods well known to those in the purification arts.
Also contemplated is sequential purification by specifically binding the antibody to a first antigen in a first purification step and to an anti-immunoglobulin antibody in a second purification step. In an alternative embodiment, the antibody is purified by sequential purification by specifically binding the antibody to a first anti-immunoglobulin antibody in a first purification step and then to a second antigen in a second purification step.
Also contemplated is sequential purification by specifically binding the antibody to a first antigen in a first purification step and to an anti-immunoglobulin antibody in a second purification step. In an alternative embodiment, the antibody is purified by sequential purification by specifically binding the antibody to a first anti-immunoglobulin antibody in a first purification step and then to a second antigen in a second purification step.
In a preferred method, sequential purification with a first and second antibody is effective in the isolation of two or more polypeptides joined by disulfide bonding comprising constant region domains which are specifically bound by a first and second antibody.
E. Diagnostic Methods
The present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of a preselected ligand, or antigen, in a sample where it is desirable to detect the presence, and preferably the amount, of the ligand,or antigen in a sample according to the diagnostic methods described herein.
In one embodiment, the antigen is progesterone or the cell surface molecules CD4 and CD3.
The sample can be a tissue, tissue extract, fluid sample or body fluid sample, such as blood, plasma or serum.
The diagnostic system includes, in an amount sufficient to perform at least one assay, a polypeptide composition according to the present invention, as a separately packaged reagent.
Exemplary diagnostic systems for detecting a preselected ligand in the solid phase and utilizing a polypeptide composition of this invention are described in the Examples.
Instructions for use of the packaged reagent(s) are also typically included.
As used herein, the term xe2x80x9cpackagexe2x80x9d refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a polypeptide of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated labeled polypeptide preparation, or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide has been operatively affixed, i.e., linked so as to be capable of binding a ligand.
xe2x80x9cInstructions for usexe2x80x9d typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
A diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of a binding reaction complex containing a ligand-binding polypeptide complexed with the preselected ligand.
The word xe2x80x9ccomplexxe2x80x9d as used herein refers to the product of a specific binding reaction such as a polypeptide-ligand reaction. Exemplary complexes are immunoreaction products.
As used herein, the terms xe2x80x9clabelxe2x80x9d and xe2x80x9cindicating meansxe2x80x9d in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed polypeptide, or phage particle that is used in a diagnostic method. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, xe2x80x9cImmunofluorescence Analysisxe2x80x9d, in Antibody As a Tool, Marchalonis, et al., eds., John Wiley and Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.
In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2xe2x80x2-amino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).
Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124I, 125I, 128I, 132I and 51Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 125I. Another group of useful labeling means are those elements such as 11C, 18F, 15O and 13N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal""s body. Also useful is a beta emitter, such 111 indium of 3H.
The linking of labels, i.e., labeling of, polypeptides and proteins or phage is well known in the art. For instance, proteins can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.
The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A xe2x80x9cspecific binding agentxe2x80x9d is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide of the present invention. Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that species is present as part of a complex.
In preferred embodiments, the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
The diagnostic kits of the present invention can be used in an xe2x80x9cELISAxe2x80x9d format to detect the quantity of a preselected ligand in a fluid sample. xe2x80x9cELISAxe2x80x9d refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample and is readily applicable to the present methods. A description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D. P. Sites et al., published by Lange Medical Publications of Los Altos, Calif. in 1982 and in U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043, which are all incorporated herein by reference.
Thus, in some embodiments, a polypeptide of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.
A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption methods are described herein.
Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, Ill.; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.
The reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme""s substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
The packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
F. Assay Methods
The present invention contemplates various assay methods for determining the presence, and preferably amount, of a preselected ligand, typically present in an aqueous composition such as a biological fluid sample using a polypeptide composition of this invention as an ligand-binding reagent to form a binding reaction product whose amount relates, either directly or indirectly, to the amount of the preselected ligand in the sample.
Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures in which a binding reagent of this invention can be used to form an binding reaction product whose amount relates to the amount of the ligand in a sample. Thus, while exemplary assay methods are described herein, the invention is not so limited.
Various heterogenous and homogeneous protocols, either competitive or noncompetitive, can be employed in performing an assay method of this invention.
In one embodiment, the invention contemplates a direct binding assay using a polypeptide composition of this invention as a binding reagent to detect the presence of a preselected ligand with which the polypeptide binds. The method comprises the steps of a) admixing a sample suspected to contain a preselected antigen with a polypeptide of this invention that binds to the preselected ligand under binding conditions sufficient for the polypeptide to bind the ligand and form a ligand-receptor complex; and b) detecting the presence of the ligand-receptor complex or the polypeptide in the complex.
Binding conditions are those that maintain the ligand-binding activity of the receptor. Those conditions include a temperature range of about 4 to 50 degrees Centigrade, a pH value range of about 5 to 9 and an ionic strength varying from about that of distilled water to that of about one molar sodium chloride.
The detecting step can be directed, as is well known in the immunological arts, to either the complex or the binding reagent (the receptor component of the complex). Thus, a secondary binding reagent such as an antibody specific for the receptor may be utilized.
Alternatively, the complex may be detectable by virtue of having used a labeled receptor molecule, thereby making the complex labeled. Detection in this case comprises detecting the label present in the complex.
A further diagnostic method utilizes the multivalency of a polypeptide composition of this invention to cross-link ligand, thereby forming an aggregation of multiple ligands and polypeptides, producing a precipitable aggregate. This embodiment is comparable to the well known methods of immune precipitation. This embodiment comprises the steps of admixing a sample with a polypeptide composition of this invention to form a binding admixture under binding conditions, followed by a separation step to isolate the formed binding complexes. Typically, isolation is accomplished by centrifugation or filtration to remove the aggregate from the admixture. The presence of binding complexes indicates the presence of the preselected ligand to be detected.
G. Therapeutic Methods
The antibodies can also be used immunotherapeutically. The term xe2x80x9cimmunotherapeuticallyxe2x80x9d or xe2x80x9cimmunotherapyxe2x80x9d as used herein in conjunction with the antibodies of the invention denotes both prophylactic as well as therapeutic administration. Thus, the antibodies can be administered to high-risk patients in order to lessen the likelihood and/or severity of disease, administered to patients already evidencing active infection, or administered to patients at risk of infection.
1. Therapeutic Compositions
The present invention therefore contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with at least one species of antibody as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a human patient for therapeutic purposes, unless that purpose is to induce an immune response.
As used herein, the terms xe2x80x9cpharmaceutically acceptablexe2x80x9d, xe2x80x9cphysiologically tolerablexe2x80x9d and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.
A therapeutic composition contains an antibody of the present invention, typically an amount of at least 0.1 weight percent of antibody per weight of total therapeutic composition. A weight percent is a ratio by weight of antibody to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of antibody per 100 grams of total composition.
2. Therapeutic Methods
In view of demonstrating T cell crosslinking and activation with bivalent antibodies of the present invention, the present disclosure provides for a method for activating and crosslinking T cells in vitro or in vivo. The method comprises contacting a sample believed to contain effector and helper T cells with a composition comprising a therapeutically effective amount of an antibody of this invention.
For in vivo modalities, the method comprises administering to the patient a therapeutically effective amount of a physiologically tolerable composition containing an antibody of the invention. Thus, the present invention describes in one embodiment a method for activating T cells in a human comprising administering to the human an immunotherapeutically effective amount of the antibody of this invention.
A representative patient for practicing the present immunotherapeutic methods is any human exhibiting symptoms of a disease which may be treated by the activation of T cells or any patient at risk for a disease which may be treated by the activation of T cells.
A therapeutically (immunotherapeutically) effective amount of an antibody is a predetermined amount calculated to achieve the desired effect, i.e., to specifically bind T cells present in the sample or in the patient, and thereby activate the T cells in the sample or patient. In the case of in vivo therapies, an effective amount can be measured by improvements in one or more symptoms associated with disease occurring in the patient, or by serological increases in the numbers of activated T cells.
Thus, the dosage ranges for the administration of the antibodies of the invention are those large enough to produce the desired effect in which the symptoms of the disease are ameliorated or the likelihood of infection decreased. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.
A therapeutically effective amount of an antibody of this invention is typically an amount of antibody such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 microgram (ug) per milliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, and usually about 5 ug/ml. Stated differently, the dosage can vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.
The antibodies of the invention can be administered parenterally by injection or by gradual infusion over time. Although the infection may be systemic and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that targeting a tissue will result in a lessening of the disease. Thus, antibodies of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be delivered by peristaltic means.
The therapeutic compositions containing an antibody of this invention are conventionally administered intravenously, as by injection of a unit dose, for example. The term xe2x80x9cunit dosexe2x80x9d when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject""s system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
As an aid to the administration of effective amounts of an antibody, a diagnostic method for detecting the antibody in the subject""s blood is useful to characterize the fate of the administered therapeutic composition.
The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising an antibody of the invention, the medicament being used for immunotherapy of a related disease.